In the last century, conversion of native North American grasslands to Juniperus virginiana forests or woodlands has dramatically altered ecosystem structure and significantly increased ecosystem carbon (C) stocks. We compared soils under recently established J. virginiana forests and adjacent native C4-dominated grassland to assess changes in potential soil nitrogen (N) transformations and plant available N. Over a 2-year period, concentrations of extractable inorganic N were measured in soils from forest and grassland sites. Potential gross N ammonification, nitrification, and consumption rates were determined using 15N isotope-dilution under laboratory conditions, controlling for soil temperature and moisture content. Potential nitrification rates (Vmax) and microbial biomass, as well as soil physical and chemical properties were also assessed. Extractable NH4+ concentrations were significantly greater in grassland soils across the study period (P ≤ 0.01), but analysis by date indicated that differences in extractable inorganic N occurred more frequently in fall and winter, when grasses were senescent but J. virginiana was still active. Laboratory-based rates of gross N mineralization (ammonification) and nitrification were greater in grassland soils (P ≤ 0.05), but only on one of four dates. Potential nitrification rates (Vmax) were an order of magnitude greater than gross nitrification rates in both ecosystems, suggesting that nitrification is highly constrained by NH4+ availability. Differences in plant uptake of N, C inputs, and soil microclimate as forests replace grasslands may influence plant available N in the field, as evidenced by seasonal differences in soil extractable NH4+, and total soil C and N accumulation. However, we found few differences in potential soil N transformations under laboratory conditions, suggesting that this grassland-to-forest conversion caused little change in mineralizable organic N pools or potential microbial activity.